Fungal infections are a significant cause of disease, degradation of quality of life, and mortality among humans, particularly for immune compromised patients. The incidence of fungal infections in humans has increased greatly in the past 20 years. This is in part due to increased numbers of people with immune systems weakened or devastated by organ transplants, cancer chemotherapy, AIDS, age, and other similar disorders or conditions. Such patients are prone to attack by fungal pathogens that are prevalent throughout the population but are kept in check by a functioning immune system. These pathogens are difficult to control because some existing antifungal agents are either highly toxic or only inhibit fungal activity. For example, the polyenes are fungicidal but toxic; whereas, the azoles are much less toxic but only fungistatic. More importantly, there have been recent reports of azole and polyene resistant strains of Candida which severely limits therapy options against such strains.
One class of new antifungal agents, the pseudomycins, shows great promise for treating fungal infections in a variety of patients. (see i.e., Harrison, L, et al., “Pseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity,” J. Gen. Microbiology, 137(12), 2857-65 (1991) and U.S. Pat. Nos. 5,576,298 and 5,837,685). Pseudomycins are natural products derived from isolates of Pseudomonas syringae. P. syringae is a large group of plant-associated bacteria that have been the source of several bioactive substances, such as bacitracin and the syringomycins. Natural strains and transposon-generated mutants of P. syringae produce compounds with antifungal activity. A transposon-generated regulatory mutant of the wild type strain of P. syringae MSU 174, known as MSU 16H (ATCC 67028), produces several pseudomycins. Pseudomycins A, B, C and C′ have been isolated, chemically characterized, and shown to possess wide spectrum antifungal activity, including activity against important fungal pathogens in both humans and plants. The pseudomycins are structurally related to but are distinct from syringomycin and other antimycotics from isolates of P. syringae. The peptide moiety for pseudomycins A, B, C, C′ corresponds to L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-OH)-L-Thr(4-Cl)SEQ ID NO:1 with the terminal carboxyl group closing a macrocyclic ring on the OH group of the N-terminal Ser. The analogs are distinguished by the N-acyl side chain, i.e., pseudomycin A is N-acylated by 3,4-dihydroxytetradeconoate, pseudomycin B by 3-hydroxytetradecanoate, pseudomycin C by 3,4-dihydroxyhexadecanoate and pseudomycin C′ by 3-hydroxyhexadecanoate. (see i.e., Ballio, A., et al., “Novel bioactive lipodepsipeptides from Pseudomonas syringae: the pseudomycins,” FEBS letters, 355(1), 96-100, (1994) and Coiro, V. M., et al., “Solution conformation of the Pseudomonas syringae MSU 16H phytotoxic lipodepsipeptide Pseudomycin A determined by computer simulations using distance geometry and molecular dynamics from NMR data,” Eur. J. Biochem., 257(2), 449456 (1998).)
Despite the increase in prevalence of fungal infections and the promise of pseudomycins, production of commercial-scale quantity pseudomycins has proved problematic. Published methods for producing pseudomycins are conducted in culture without agitation or stirring and employing expensive potato dextrose medium. Such cultures typically produce pseudomycins in small amounts suitable for laboratory studies and concentrations that are unsuitable for production on a large or commercial scale. Still culture is also unsuitable for use on a scale of greater than about one liter where diffusion of nutrients, gases, especially oxygen, and maintaining adequate cell growth become problematic.
There remains a need for a method for producing pseudomycins using P. syringae on a scale and at a concentration suitable for commercial uses of the pseudomycins as antifungal agents for application against infections in animals, humans, or plants.